A short peptide within Nsa2 is essential for binding to Rsa4. (A) In vitro binding assay of MBP-Nsa2 with Rsa4. Purified MBP-Nsa2 (lane 4–6), MBP-Nsa2 84–96 aa (lane 7–9), and MBP (lane 10–12) were immobilized on amylose beads and incubated with E. coli lysate without or with HIS₆-TEV-Rsa4 full-length or HIS₆-Rsa4 WD40 domain, respectively (lane 1–3). Eluates were analyzed by 4–12% SDS-PAGE and stained with Coomassie blue. (B) In vitro copurification of GST-Nsa2 and Rsa4. GST-Nsa2 wild-type, GST-Nsa2Δ86–90, or GST-Nsa2Δ85–98 were coexpressed with HIS-Rsa4 (input; see lanes 1, 2, and 3), purified via GSH Sepharose, and eluted by TEV cleavage (lanes 5, 6, and 7). The 4–12% SDS-polyacrylamide gel was stained with Coomassie blue, and Rsa4 and Nsa2 were detected by Western blot analysis. (C) Viability of nsa2 deletion mutants. An Nsa2 shuffle strain was transformed with plasmids encoding the indicated NSA2 alleles tagged with FTpA. Transformants were analyzed for complementation by spotting a 1:10 dilution series on SDC+FOA. The growth phenotype after 3 d of incubation at 30°C is shown. (D) Dominant-negative phenotype of nsa2 deletion mutants. Wild-type strain W303 was transformed with plasmids expressing the indicated NSA2 alleles tagged with FTpA under the control of the inducible GAL10 promoter. The toxic effect of NSA2 overexpression was tested on galactose-containing medium (SGC-TRP) after incubation for 3 d at 30°C. (E) ITC measurement of Rsa4 β-propeller with Nsa2 peptide is shown. Recombinant Rsa4 β-propeller (Rsa4Δ136) was expressed in E. coli, affinity-purified, and further purified by SEC before ITC measurement was performed with synthesized Nsa2 peptide (85–95 aa, DALPTYLLDRE).